Methods for detecting and quantifying binding and inhibition of binding of species to nucleic acids

Abstract

The invention features methods for detecting and quantifying the binding or inhibition of binding of species to biopolymers, e.g., nucleic acids. The invention is based on the use of probes that have magnetic relaxation properties that are affected by the presence of paramagnetic metal ions, e.g., Mn 2+ . Any class of biopolymer that binds metal ions at its active site or uses metal ion cofactors can be studied using these methods.

1. A method of detecting the binding of a species to a nucleic acid, said method comprising the steps of:
 (a) measuring a magnetic relaxation property of a probe in a first solution using nuclear magnetic resonance spectroscopy, wherein said first solution comprises said nucleic acid, a first concentration of said species, a paramagnetic metal ion, and said probe; 
 (b) measuring the magnetic relaxation property of said probe in a second solution using nuclear magnetic resonance spectroscopy, wherein said second solution comprises said nucleic acid, a second concentration of said species, a paramagnetic metal ion, and said probe; and 
 (c) comparing the relaxation property measured in step (a) with the relaxation property measured in step (b), wherein a difference in said relaxation properties indicates the binding of said species to said nucleic acid. 
 
     
     
       2. The method of  claim 1 , wherein, in step (c), said magnetic relaxation properties of said probe are correlated with said first and second concentrations of said species, thereby quantifying the binding of said species to said nucleic acid. 
     
     
       3. The method of  claim 1 , wherein said first concentration is 0.0 μM. 
     
     
       4. The method of  claim 1 , wherein said species is a divalent metal ion. 
     
     
       5. The method of  claim 4 , wherein said metal ion is Mg 2+ . 
     
     
       6. The method of  claim 1 , wherein said species is a candidate therapeutic agent. 
     
     
       7. The method of  claim 6 , wherein said candidate therapeutic agent is selected from a molecular library. 
     
     
       8. The method of  claim 1 , wherein said species is selected from the group consisting of a protein, a peptide, and a fragment of a protein. 
     
     
       9. The method of  claim 1 , wherein said paramagnetic metal ion is selected from the group consisting of Mn 2+ , Fe 2+ , Fe 3+ , Co 2+ , Ni 2+ , or a lanthanide ion. 
     
     
       10. The method of  claim 1 , wherein said paramagnetic metal ion is bound to said nucleic acid. 
     
     
       11. The method of  claim 1 , wherein said probe comprises an X—H bond, wherein X is an NMR-active nucleus. 
     
     
       12. The method of  claim 11 , wherein X is  31 P. 
     
     
       13. The method of  claim 12 , wherein said probe is methylphosphite, ethylphosphite, or ethylphosphonite. 
     
     
       14. The method of  claim 11 , wherein said magnetic relaxation property of X is indirectly detected using X edited,  1 H detected NMR spectroscopy. 
     
     
       15. The method of  claim 14 , wherein said measuring of said magnetic relaxation properties of X proceeds by the steps of:
 (i) using a pulse sequence to transfer coherent magnetization originating on the  1 H nucleus to the X nucleus by Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) techniques prior to a T 2  delay; 
 (ii) providing a T 2  delay; and 
 (iii) transferring the remaining coherence back to the  1 H nucleus for detection using a reverse INEPT series of pulses. 
 
     
     
       16. The method of  claim 1 , wherein said magnetic relaxation property is the T 2  relaxation of a nucleus of said probe. 
     
     
       17. The method of  claim 1 , wherein said nucleic acid comprises RNA. 
     
     
       18. The method of  claim 1 , wherein said nucleic acid comprises DNA. 
     
     
       19. The method of  claim 1 , wherein said nucleic acid is double-stranded. 
     
     
       20. A method of detecting the binding of a paramagnetic metal ion to a nucleic acid, said method comprising the steps of:
 (a) measuring a magnetic relaxation property of a probe in a first solution using nuclear magnetic resonance spectroscopy, wherein said first solution comprises a first concentration of said paramagnetic metal ion, said nucleic acid, and said probe; 
 (b) measuring the magnetic relaxation property of said probe in a second solution using nuclear magnetic resonance spectroscopy, wherein said second solution comprises a second concentration of said paramagnetic metal ion, said nucleic acid, and said probe; and 
 (c) comparing the relaxation property measured in step (a) with the relaxation property measured in step (b), wherein a difference in said relaxation properties indicates the binding of said paramagnetic metal ion to said nucleic acid. 
 
     
     
       21. The method of  claim 20 , wherein, in step (c), said magnetic relaxation properties of said probe are correlated with said first and second concentrations of said nucleic acid, thereby quantifying the binding of said paramagnetic metal ion to said nucleic acid. 
     
     
       22. The method of  claim 20 , wherein said first concentration is 0.0 μM. 
     
     
       23. The method of  claim 20 , wherein said paramagnetic metal ion is selected from the group consisting of Mn 2+ , Fe 2+ , Fe 3+ , Co 2+ , Ni 2+ , or a lanthanide ion. 
     
     
       24. The method of  claim 1 , wherein said probe comprises an X—H bond, wherein X is an NMR-active nucleus. 
     
     
       25. The method of  claim 18 , wherein X is  31 P. 
     
     
       26. The method of  claim 25 , wherein said probe is methylphosphite, ethylphosphite, or ethylphosphonite. 
     
     
       27. A method of determining the effect of a first species on the binding of a second species to a nucleic acid, said method comprising the steps of:
 (a) measuring a magnetic relaxation property of a probe in a first solution using nuclear magnetic resonance spectroscopy, wherein said first solution comprises a first concentration of said first species, said nucleic acid, said second species that binds to said nucleic acid, a paramagnetic metal ion, and a probe; 
 (b) measuring the magnetic relaxation property of said probe in a second solution using nuclear magnetic resonance spectroscopy, wherein said second solution comprises a second concentration of said first species, said nucleic acid, said second species, said paramagnetic metal ion, and said probe; and 
 (c) comparing the relaxation property measured in step (a) with the relaxation property measured in step (b), wherein a difference in said relaxation properties indicates the inhibition of the binding of said second species to said nucleic acid. 
 
     
     
       28. The method of  claim 27 , wherein, in step (c), said magnetic relaxation properties of said probe are correlated with said first and second concentrations of said first species, thereby quantifying the effect of said first species on the binding of said second species to said nucleic acid. 
     
     
       29. The method of  claim 27 , wherein said first concentration is 0.0 μM. 
     
     
       30. The method of  claim 27 , wherein one of said first species and said second species is a divalent metal ion. 
     
     
       31. The method of  claim 30 , wherein said metal ion is Mg 2+ . 
     
     
       32. The method of  claim 27 , wherein said first species or said second species is a candidate therapeutic agent. 
     
     
       33. The method of  claim 32 , wherein candidate therapeutic agent is selected from a molecular library. 
     
     
       34. The method of  claim 27 , wherein said first or said second species is selected from the group consisting of a protein, a peptide, and a fragment of a protein. 
     
     
       35. The method of  claim 27 , wherein said paramagnetic metal ion is selected from the group consisting of Mn 2+ , Fe 2+ , Fe 3+ , Co 2+ , Ni 2+ , or a lanthanide ion. 
     
     
       36. The method of  claim 27 , wherein said paramagnetic metal ion is bound to said nucleic acid. 
     
     
       37. The method of  claim 27 , wherein said probe comprises an X—H bond, wherein X is an NMR-active nucleus. 
     
     
       38. The method of  claim 27 , wherein X is  31 P. 
     
     
       39. The method of  claim 38 , wherein said probe is methylphosphite, ethylphosphite, or ethylphosphonite. 
     
     
       40. A method of detecting the availability of a paramagnetic metal ion in a solution comprising a nucleic acid, said method comprising the steps of:
 (a) providing a solution comprising said nucleic acid, a probe, and said paramagnetic metal ion and 
 (b) measuring a magnetic relaxation property of said probe in said solution using nuclear magnetic resonance spectroscopy, wherein the magnitude of said magnetic property is indicative of the availability of said paramagnetic ion in said solution. 
 
     
     
       41. The method of  claim 40 , wherein said paramagnetic metal ion is bound to said nucleic acid.